Preservation solutions

ABSTRACT

There is described a preservation solution for the preservation of cells, tissues and/or organs, said solution comprising: (i) water for injection; (ii) at least one saccharide; (iii) at least one component with pH buffer properties; (iv) optionally at least one component with calcium transport blocking properties or an anti-calcium action activity; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine; provided that acetamide is absent and/or if aspirin is present glutamine is absent and if glutamine is present aspirin is absent.

FIELD OF THE INVENTION

The present invention relates to a preservation solution or a coldstorage preservation solution to keep cells without a blood supplyviable, the use thereof to prevent damage to cells, tissues and/ororgans in transplantation, surgery, experimentally and in vitro, amethod for preservation, flush or flush preservation, a method fortreatment, a method for preparation thereof, and a kit of partscomprising the solution.

More particularly the present invention relates to a preservationsolution or a cold storage preservation solution as herein describedwhich comprises salicylic acid and/or glutamic acid; and salts thereof.

BACKGROUND TO THE INVENTION

Organ transplantation is now available for kidney, liver, heart, lung,pancreas and intestine. At retrieval a transplant organ is flushedthrough its vasculature with a preservation solution. This solution isdesigned to facilitate the reduction of temperature of the organ,prevent cell swelling, remove oxygen free radicals, control pH, reduceischaemic damage, extend the safe time for which organs can be kept outof the body and facilitate recovery of the organ upon reperfusion.

Important flush solutions were introduced by Belzer in 1967 and Collinsin 1969, subsequently modified to Euro-Collins (EC), Marshall (1976),Bretschneider (see Isemer et al 1988), and others. University ofWisconsin solution (UW), the most successful of all solutions, wasintroduced in 1988 by Belzer and his colleagues. There remains a needfor improved flush preservation and cold storage preservation.Overwhelming evidence indicates that a high quality graft provides bothbetter immediate function and a longer functional graft lifetime.

A simple flush solution containing only sodium phosphate and sucrose wasshown by Andrews and Coffey in 1982, and by Coffey and Andrews in 1983to protect the morphology of kidney tubules from ischaemic damage.

International patent application No. WO 02/41696 (Potts, Lodge)describes a flush preservation solution for the preservation of cells inthe absence of a blood supply, wherein said solution comprises:

-   -   water for injection;    -   at least one saccharide, such as a monosaccharide, disaccharide,        trisaccharide, or polysaccharide;    -   at least one component with pH buffer properties (i.e. a pH        buffer);    -   at least one component with calcium transport blocking        properties or an anti-calcium action activity (i.e. a calcium        transport blocker);    -   optionally an amino acid, such as glutamine; and    -   optionally a thromboxane inhibitor to prevent blood clotting,        such as aspirin.

Accordingly, there remains a need for an improved commercially viableand effective preservation solution which enables extended preservationof cells, tissues and organs, including engineered cells, tissues andorgans, which provides improved versatility, effectiveness andreperfusion in transplantation, in surgery, including any situation ofwarm or cold ischaemia, such as transplantation of liver, kidney, smallbowel and/or pancreas; whole limb, whole body, or in experimentation.

SUMMARY OF THE INVENTION

We have found that the aspirin (aspirin is the common name foracetylsalicylic acid) in the prior art solution of WO 02/41696 degradesto produce salicylic acid, whilst the glutamine degrades to glutamicacid.

Degradation of aspirin will produce salicylic acid and acetic acid,whilst degradation of glutamine will produce glutamic acid and ammonia.Moreover, we have found that both acetic acid and ammonia areundesirable in a preservation solution as they are both potentiallydamaging to cells, tissues and organs; and are likely to generatenoxious odours, which may increase on storage of the preservationsolution. Furthermore, we have found that over time acetic acid andammonia may react together in the prior art solution of WO 02/41696, toform acetamide. Acetamide is recognised as a skin irritant and isrecognised as a hazardous substance which may damage the liver.

Thus, by replacing aspirin with salicylic acid and/or replacingglutamine with glutamic acid, or salts thereof, the amount of aceticacid and/or ammonia respectively generated, may be minimised or removedcompletely. In examples, it is demonstrated that the use of the solutionof the invention for preservation reduces damage to cells and tissuesduring preservation when compared to comparative solutions.

In the prior art solution, aspirin is added as an anti-inflammatorythromboxane inhibitor. We have found that if the aspirin is omitted andsalicylic acid added, salicylic acid has anti-inflammatory properties(i.e. is an anti-inflammatory). Also, in the prior art solution,glutamine is added as a supporting molecule which can provide energy. Wehave found that if glutamine is omitted it can be replaced by glutamicacid which will act as an alternative energy substrate for the cells,tissues and/or organs.

In addition, we have found that since the novel solution of the presentinvention no longer represents any concern about the degradation ofaspirin and/or glutamine, the amounts of the replacement components,e.g. salicylic acid and/or glutamic acid can consequently be reduced.

Therefore, according to a first aspect of the invention there isprovided a preservation solution for the preservation of cells, tissuesand/or organs, in the absence of a blood supply, said solutioncomprising:

-   -   (i) water for injection;    -   (ii) at least one saccharide, such as a monosaccharide,        disaccharide, trisaccharide, or polysaccharide;    -   (iii) at least one component with pH buffer properties;    -   (iv) optionally at least one component with calcium transport        blocking properties or an anti-calcium action activity;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine;    -   provided that acetamide is absent and/or if aspirin is present        glutamine is absent and if glutamine is present aspirin is        absent.

The term “absent” or “substantially absent” shall be understood by theperson skilled in the art to mean below a pharmacologically activeconcentration; or none detectable by conventional methods known in theart. Such methods shall include, but shall not be limited tocolorimetric assay and GCMS.

As a further proviso, if only one of aspirin and glutamine is present,then the degradation product of the other is not present, that is tosay, if aspirin is present then ammonia is not present; and if glutamineis present then acetic acid is not present.

Salicylic acid, in free form or in salt form, or aspirin, (v) suitablyserves to prevent blood clotting. Suitably the preservation solutioncomprises an amount of salicylic acid, in free form or in salt form, oraspirin (v) to prevent blood clotting.

Glutamic acid, in free form or in salt form, or glutamine, (vi) suitablyserves as an energy substrate. Suitably the preservation solutioncomprises an amount of glutamic acid, in free form or in salt form, orglutamine (vi) to serve as an energy substrate.

In one particular aspect of the present invention the preservationsolution as herein described comprises:

-   -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine;    -   provided that acetamide is absent and/or at least one of acetic        acid and ammonia is absent.

According to a further aspect of the present invention there is provideda preservation solution for the preservation of cells, tissues and/ororgans, said solution comprising:

-   -   (i) water for injection;    -   (ii) at least one saccharide, such as a monosaccharide,        disaccharide, trisaccharide, or polysaccharide;    -   (iii) at least one component with pH buffer properties;    -   (iv) optionally at least one component with calcium transport        blocking properties or an anti-calcium action activity;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine;    -   wherein if (v) is aspirin (vi) is glutamic acid, in free form or        salt form, and if (vi) is glutamine (v) is salicylic acid, in        free form or salt form.

Preferably the preservation solution comprises an admixture of (i) to(vi) as herein described, thereby providing said preservation solutionfrom which acetamide is absent. For example, one of aspirin andglutamine is present and acetamide is absent and/or one of acetic acidand ammonia is present and acetamide is absent, or both of aspirin andglutamine, and both of acetic acid and ammonia are absent, and acetamideis absent.

In one aspect of the invention there is provided a preservation solutionas herein described for the preservation of cells, tissues and/ororgans, in the absence of a blood supply.

In embodiments herein there is provided a prepared preservation solutioncomprising a preservation solution as herein described, whereinacetamide is absent, for example one of aspirin and glutamine is presentand acetamide is absent and/or one of acetic acid and ammonia is presentand acetamide is absent, or both of aspirin and glutamine, and both ofacetic acid and ammonia are absent, and acetamide is absent.

The prepared preservation solution may be packaged for storage, e.g.under anoxic conditions, in the absence of UV light. The preparedpreservation solution herein described is sterile and packaged forstorage under anoxic conditions in the absence of UV light.

According to one aspect of the invention the preservation solution is aflush preservation solution.

According to another aspect of the invention the preservation solutionis a cold storage preservation solution.

In one embodiment, (v) is salicylic acid, in free form or in salt form;and

-   -   (vi) is glutamic acid, in free form or in salt form, or        glutamine; provided that aspirin is absent, preferably aspirin        and acetamide are absent and/or acetic acid and acetamide are        absent.

In another embodiment, (v) is salicylic acid, in free form or in saltform, or aspirin; and

-   -   (vi) is glutamic acid, in free form or in salt form;

provided that glutamine is absent, preferably glutamine and acetamideare absent and/or ammonia and acetamide are absent.

In another embodiment, (v) is salicylic acid, in free form or in saltform; and

-   -   (vi) is glutamic acid, in free form or in salt form;

provided that glutamine and aspirin are absent, preferably acetamide isadditionally absent and/or acetic acid, ammonia and acetamide areabsent.

In a preferred preservation solution of the invention, aspirin isreplaced by salicylic acid, in free form or in salt form, and glutamineis replaced by glutamic acid, in free form or in salt form, such thatboth aspirin and glutamine are substantially absent from the solution,preferably acetamide is additionally absent and/or acetic acid, ammoniaand acetamide are absent.

In another embodiment of the invention there is provided a preservationsolution as herein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and/or        aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and/or        glutamine;

provided that if aspirin is present glutamine is absent and if glutamineis present aspirin is absent and/or wherein if aspirin and/or acetamideand/or acetic acid is present, the amount of salicylic acid presentexceeds that which can be attributed to degradation from aspirin, forexample as determined by the sum of the amounts of any acetic acid andof any acetamide present; and/or

-   -   wherein if glutamine and/or acetamide and/or ammonia is present,        the amount of glutamic acid present exceeds that which can be        attributed to degradation from glutamine, for example as        determined by the sum of the amounts of any ammonia and of any        acetamide present.

In particular, there is provided a preservation solution as hereindescribed comprising:

-   -   (v) salicylic acid, in free form or salt form, and/or aspirin;        and    -   (vi) glutamic acid, in free form or salt form, and/or glutamine;        provided that (v) is salicylic acid, in free form or salt form,        and aspirin and/or

(vi) is glutamic acid, in free form or salt form, and glutamine;

-   -   provided that if aspirin is present glutamine is absent and if        glutamine is present aspirin is absent; and/or        -   wherein if aspirin and/or acetamide and/or acetic acid is            present, the amount of salicylic acid present exceeds that            which can be attributed to degradation from aspirin, for            example as determined by the sum of the amounts of any            acetic acid and of any acetamide present; and/or        -   wherein if glutamine and/or acetamide and/or ammonia is            present, the amount of glutamic acid present exceeds that            which can be attributed to degradation from glutamine, for            example as determined by the sum of the amounts of any            ammonia and of any acetamide present.

There is also provided a preservation solution as herein describedcomprising:

-   -   (v) salicylic acid, in free form or in salt form, and optionally        additionally aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and/or        glutamine;    -   provided that acetamide is absent and/or if aspirin is present        glutamine is absent and if glutamine is present aspirin is        absent; or        -   wherein if aspirin and/or acetamide and/or acetic acid is            present, the amount of salicylic acid present exceeds that            which can be attributed to degradation from aspirin, for            example as determined by the sum of the amounts of any            acetic acid and of any acetamide present; and/or        -   wherein if glutamine and/or acetamide and/or ammonia is            present, the amount of glutamic acid present exceeds that            which can be attributed to degradation from glutamine, for            example as determined by the sum of the amounts of any            ammonia and of any acetamide present.

There is further provided a preservation solution as herein describedcomprising:

-   -   (v) salicylic acid, in free form or in salt form, and optionally        additionally aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and/or        glutamine;

provided that: when (v) is salicylic acid, in free form or in salt formand aspirin is absent, (vi) is glutamic acid, in free form or in saltform, and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and optionally        additionally aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, and/or        glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form; and    -   (vi) glutamic acid, in free form or in salt form and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and/or        aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and optionally        additionally glutamine;        -   provided that acetamide is absent and/or if aspirin is            present glutamine is absent and if glutamine is present            aspirin is absent;        -   or wherein if glutamine and/or acetamide and/or ammonia is            present, the amount of glutamic acid present exceeds that            which can be attributed to degradation from glutamine, for            example as determined by the sum of the amounts of any            ammonia and of any acetamide present and/or        -   wherein if aspirin and/or acetamide and/or acetic acid is            present, the amount of salicylic acid present exceeds that            which can be attributed to degradation from aspirin, for            example as determined by the sum of the amounts of any            acetic acid and of any acetamide present.

According to this aspect of the invention there is provided apreservation solution as herein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and/or        aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and optionally        additionally glutamine;    -   provided that: when (vi) is glutamic acid, in free form or in        salt form and glutamine is absent, (v) is salicylic acid, in        free form or in salt form, and aspirin.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, and optionally        additionally glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and/or        aspirin; and    -   (vi) glutamic acid, in free form or in salt form, and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, and glutamine.

According to the invention there is provided a preservation solution asherein described comprising:

-   -   (v) salicylic acid, in free form or in salt form, and aspirin;        and    -   (vi) glutamic acid, in free form or in salt form.

For the avoidance of doubt it will be understood by the person skilledin the art that reference herein to cells, tissues and organs, shallinclude engineered cells, tissues and organs.

In one aspect of the invention at least one component with calciumtransport blocking properties or an anti-calcium action activity ispresent in the preservation solution of the invention.

In another aspect of the invention the component with calcium transportblocking properties or an anti-calcium action activity is absent fromthe preservation solution of the invention.

In one aspect in the preservation solution of the invention the amountof salicylic acid, in free form or in salt form, sufficient to preventblood clotting, is less than 0.5 mmol/L, for example from about 0.025 toless than 0.3 mmol/L. Alternatively, the amount of salicylic acidsufficient to prevent blood clotting, is from about 0.025 to about 0.275mmol/L, preferably about 0.25 mmol/L salicylic acid, in free form or insalt form.

Alternatively, aspirin, if present, is suitably present in an amount offrom about 0.3 mmol/L to about 1.0 mmol/L, for example 0.5 mmol/L.

In another aspect in the preservation solution of the invention theamount of glutamic acid, in free form or in salt form, present is lessthan 20 mmol/L. Alternatively, the amount of glutamic acid present isfrom about 2 to less than 15, or from about 15 to less than 20, such asfrom about 2 to about 12 mmol/L, preferably about 3 mmol/L glutamicacid, in free form or in salt form.

Alternatively glutamine, if present, is suitably present in an amount offrom about 15 mmol/L to about 30 mmol/L for example approximately 20mmol/L. Alternatively, the amount of glutamine present is from about 2to less than 15 mmol/L.

The salts of salicylic acid and/or glutamic acid, which may be the sameor different, will be pharmacologically acceptable salts, that is, saltsthat possesses the desired pharmacological activity of the parentcompound. In particular, such salts shall be non-toxic to cells, tissuesand/or organs. Such salts will generally be formed when an acidic protonpresent in the salicylic acid and/or glutamic acid either is replaced bya metal ion, e.g., an alkali metal ion or an alkaline earth ion. By wayof example only, alkali metal salts include sodium or potassium, and thelike. By way of example only, alkaline earth metal salts include calciumor magnesium, and the like.

The flush solution may consist only of these components, in which caseit is suited for preservation of universal cell types and functioning,in particular for preservation of simple cell systems, alternatively itmay be provided together with one or more further substituentsspecifically suited to the preservation of a desired type or function ofcell, in particular in the preservation of complex cell systems such asorgans or living tissue, more particularly for small or large animals,most particularly human organs, such as liver, kidney, small boweland/or pancreas; and living tissue.

According to this aspect of the invention there is provided apreservation solution for liver, kidney, small bowel and pancreaspreservation comprising a combination of

-   -   (i) water for injection;    -   (ii) a disaccharide;    -   (iii) at least one component with pH buffer properties;    -   (iv) at least one calcium transport or channel blocker;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine,        as hereinbefore defined; together with an impermeant        sequestering anion, inorganic solutes, components effective        against oxygen free radicals and a colloidal osmotic, and any        optional additional components as hereinbefore defined.

There is further provided a preservation solution for liver, kidney,small bowel and pancreas preservation comprising a combination of

-   -   (i) water for injection;    -   (ii) sucrose;    -   (iii) Na₂HPO₄ and NaH₂PO₄;    -   (iv) diltiazem;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine,        as hereinbefore defined; together with lactobionic acid, KOH and        NaOH, glutathione and allopurinol and PEG, and any optional        additional components as hereinbefore defined.

The preservation solution of the present invention has a number ofadvantages in terms of improving existing solutions, with reduced damageduring preservation, due the absence of toxic ammonia and acetic acid,and the possibility to extend preservation periods, in addition to theprovision of a kit from which to create a particular desired solution,with the associated convenience and cost implications which will rendersuch solution commercially viable.

The present invention has found that the preservation solution definedherein is universally acceptable, based on experiments and withoutattempting to rationalise the underlying preservation mechanism.

All components of the preservation solution of the present inventionsatisfy National or International Pharmacopoeial Standards of puritywhere applicable. Water for injection is typically purified andde-ionized prior to sterilization.

A saccharide is selected from sucrose, raffinose and mannitol; andcombinations thereof. A preferred saccharide is sucrose. A saccharidemay be present in an amount of from about 50 mmol/L to about 150 mmol/L,for example, about 100 mmol/L.

A pH buffer is selected from a sodium phosphate buffer, a potassiumphosphate buffer and the like, preferably Na₂HPO₄, NaH₂PO₄, K₂HPO₄,KH₂PO₄ and the like; and combinations thereof. A pH buffer may bepresent in an amount of from about 15 mmol/L to about 75 mmol/L, forexample, from about 15 mmol/L to about 20 mmol/L or from about 40 toabout 70 mmol/L.

The preservation solution of the invention is preferably formulated tocomply with a desired range of the pharmacopoeially acceptable physicalproperties. Preferably the solution has a pH in the range 6.5-7.8, morepreferably 6.5-7.0, most preferably 6.8-7.0. The pH is generallymeasured at room temperature, e.g. about 20° C.

An optionally added calcium transport blocker or anti-calcium activityagent is selected from any known calcium transport or channel blocker,such as nicardipine, diltiazem, verapamil, nisoldipine, chlorpromazineor trifluorperazine; and combinations thereof and metabolites thereof.Preferably a calcium transport or channel blocker is nicardipine and/ordiltiazem; or metabolites thereof. A calcium transport or channelblocker may be present in an amount of from about 0.0005 mmol/L to about1.0 mmol/L, for example, about 0.005 mmol/L. When the calcium transportor channel blocker is nicardipine, nicardipine may be present in anamount of from about 0.0005 mmol/L to about 1.0 mmol/L, for example,about 0.005 mmol/L. When the calcium transport or channel blocker isdiltiazem, diltiazem may be present in an amount of from 0.0005 mmol/Lto about 1.0 mmol/L, for example, about 0.022 mmol/L.

Without being limited to this theory, reference is made herein tocomponents by function, based on commonly accepted pharmacological andphysiological activity, however for the avoidance of doubt, componentslisted may contribute additional or different function to thatattributed, and this should not be seen as a limitation thereof.Additionally, functional equivalents to those listed may be consideredwithin the scope of this invention.

Other components may be present in the preservation solution. Othercomponents, unless otherwise indicated, will typically be present inminor amounts for example in the range up to 1 mmol/L.

Preferably, the preservation solution of the invention comprises one ormore additional components selected from:

-   -   at least one anion that is largely impermeable into cells,        preferably is an impermeant sequestering anion; and    -   at least one component with colloid osmotic properties (i.e. a        colloidal osmotic).

An impermeant sequestering anion preferably comprises lactobionate orlactobionic acid. When an impermeant sequestering anion is present, itmay be in an amount of from about 15 mmol/L to about 75 mmol/L, forexample, from about 15 mmol/L to about 20 mmol/L or from about 40 mmol/Lto about 70 mmol/L.

A colloidal osmotic is preferably selected from polyethylene glycol(PEG), succinylated gelatin (as in Gelofusine), Ficoll (apolysaccharide) and a starch product; and combinations thereof. When acomponent with colloid osmotic properties is present, it may be in anamount of from about 0.5 mmol/L to about 3.0 mmol/L, for example fromabout 0.75 mmol/L to about 1.33 mmol/L, such as, about 1.0 mmol/L, and20,000 MW.

Alternatively or additionally, the preservation solution of theinvention may comprise one or more components selected from:

-   -   inorganic or organic solutes;    -   a component or components with calcium chelating properties        (i.e. a calcium chelator); and    -   a component or components with iron chelating properties (i.e.        an iron chelator).

Preferably an inorganic or organic solute comprises an inorganic soluteand is an electrolyte including cations and/or anions, for exampleselected from Na⁺, K⁺, Cl⁻, OH⁻, and the like; and combinations thereof.In a preferred aspect of the invention the solute is an inorganicsolute, such as NaCl. When an inorganic or organic solute is present, itmay be in an amount of from about 15 mmol/L to about 75 mmol/L, forexample, from about 15 mmol/L to about 20 mmol/L or from about 40 mmol/Lto about 70 mmol/L.

Preferably a calcium chelator comprises citrate or EGTA (ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid) and an ironchelator comprises EDTA (ethylene diamine tetra acetic acid).

Alternatively, or additionally, the preservation solution of theinvention may comprise one or more components selected from:

-   -   one or more (additional) amino acids, in addition to glutamic        acid and/or glutamine;    -   at least one component that is effective against oxygen free        radicals or the production of oxygen free radicals;    -   at least one component of the energy supply system or which        influences the energy supply system or a ketone body; and    -   at least one component that has a cryoprotectant action.

Preferably, an additional amino acid is selected from glycine andn-acetylcysteine, and a combination thereof. When an additional aminoacid component is present, it may be in an amount of from about 1 mmol/Lto about 30 mmol/L, preferably from about 3 mmol/L to about 12 mmol/L,for example about 10 mmol/L.

Preferably oxygen free radical inhibitors are selected from allopurinoland reduced glutathione, more preferably a combination thereof. When acomponent effective against oxygen free radicals is present, it may bein an amount of from about 0.2 mmol/L to about 5 mmol/L, for example,from about 3 mmol/L to about 5 mmol/L. Generally, the amount of oxygenfree radical inhibitors defined herein refers to the total amount ofoxygen free radical inhibitors. For example, when the oxygen freeradical inhibitors comprises a combination of allopurinol and reducedglutathione, the amount of the combined oxygen free radical inhibitorsmay be in an amount of from about 0.2 mmol/L to about 5 mmol/L, etc.

Preferably an energy supply system component comprises adenosine. Whenan energy supply component is present, it may be in an amount of up toabout 20 mmol/L, for example, from about 5 to about 20 mmol/L, such asabout 5 mmol/L.

Preferably a ketone body comprises beta-hydroxy butyrate.

Cryoprotectants are compounds that when present in solution can reduceor inhibit ice crystal formation in solutions exposed to sub 0° C.temperatures. Thus, the use of a cryoprotectant enables the cells,tissue or organ to be stored at a temperature of from about −20° C. toabout 4° C. Preferably a cryoprotectant is selected from a glycol suchas propylene glycol, DMSO, a saccharide, a carbohydrate, a lipid, aglycolipid such as xylomannan, a glycoprotein, protein or polypeptide ora polyol; or a combination thereof. When a cryoprotectant is present, itmay be in an amount of from about 5 to about 100 mg/mL.

Alternatively, or additionally, the preservation solution of theinvention may comprise additional components for a specific functionselected from:

-   -   at least one component of the intracellular signal transduction        system or which modifies this system, preferably a protein        kinase inhibitor or a calmodulin inhibitor; and    -   at least one component that has a membrane stabilising action,        preferably ranolazine, and the like.

Preferably, a saccharide component (ii) is present in an amount in therange 50-150 mmol/L, for example, approximately 100 mmol/L;

-   -   the pH buffer component (iii) is present in a total amount in        the range 15-75 mmol/L, for example approximately 15-20 or 40-70        mmol/L;    -   the an impermeant sequestering anion is present in a total        amount in the range 15-75 mmol/L, for example approximately        15-20 or 40-70 mmol/L;    -   the inorganic or organic solute component is present in a total        amount in the range 15-75 mmol/L, for example approximately        15-20 or 40-70 mmol/L;    -   the additional amino acid component is present in an amount in        the range 5-30 mmol/L, preferably 5-12 mmol/L, for example        approximately 10 mmol/L;    -   the component effective against oxygen free radicals is present        in a total amount in the range up to 5 mmol/L, for example        approximately 3-5 mmol/L;    -   the energy supply component is present in an amount in the range        of up to 20 mmol/L, for example 5 mmol/L;    -   the component with colloid osmotic properties is present in an        amount in the range 0.5-3.0 mmol/L, for example approximately        0.75-1.33 mmol/L, such as 1.0 mmol/L, and 20,000 MW;

when the component with calcium transport blocking properties (iv) ispresent, the calcium transport blocking agent may be, for example,nicardipine or diltiazem. Nicardipine may be present in an amount in therange 0.0005-1.0 mmol/L, for example, 0.005 mmol/L; and diltiazem may bepresent in an amount in the range 0.0005-1.0 mmol/L, for example, 0.022mmol/L.

In the case of certain components present as 2 or more types, therelative amounts may be critical or non-critical. A preferred componenteffective against oxygen free radicals is approximately 3 mmol/L reducedglutathione and 0.35 to 0.4, more preferably 0.4 mmol/L allopurinol.

A preferred inorganic or organic solute component comprises electrolytesas follows:

-   -   Na⁺ 15-150 mmol/L, e.g. 30 mmol/L    -   K⁺ 0-25 mmol/L, e.g. 15 mmol/L    -   Cl⁻ 0-100 mmol/L    -   OH⁻ 0-75 mmol/L

Preferably a solution according to the invention is prepared and storedunder anoxic condition in the absence of UV light.

It is a particular advantage that the solution as defined comprisingcomponents (i)-(vi) may be stored for extended periods, and additionalcomponents required for clinical use (e.g. heparin) added immediatelyprior to use thereof. It is especially an advantage of the solution ofthe present invention that the solution may be stored for extendedperiods without the generation of undesirable degradation products. Moreparticularly, the solution may be stored without the generation of oneor both of acetic acid and ammonia, and without the generation ofacetamide.

Alternatively, the solution may be stored with the generation of one orboth of acetic acid and ammonia, and/or with the generation ofacetamide, in amount less than the molar equivalent of salicylic acidand glutamic acid respectively.

The preservation solution of the invention as herein defined preferablycomprises the basic components (i)-(vi) together with additionalcomponents for specific function. The solution for use in preservingorgans is particularly of greater complexity than that for preservingsimple cell systems, however we have found that the solution maynevertheless be relatively straightforward.

Preferably a preservation solution for intra-abdominal organs such askidney, liver and pancreas; and also intestine and bowel and the like;comprises components (i)-(vi) as herein defined together with at leastone component selected from an impermeant sequestering anion component;and a component with colloid osmotic properties; and more preferably,additionally one or more components selected from:

-   -   inorganic or organic solute component;    -   an additional amino acid component;    -   a component effective against oxygen free radicals; and    -   an energy supply component.

Preferably a preservation solution for liver, kidney, small bowel andpancreas preservation comprises a combination:

-   -   (i) water for injection;    -   (ii) a disaccharide;    -   (iii) at least one component with pH buffer properties;    -   (iv) at least one calcium transport or channel blocker;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine,        as hereinbefore defined; together with an impermeant        sequestering anion, inorganic solutes, components effective        against oxygen free radicals and a colloidal osmotic, and any        optional additional components as herein defined;    -   more preferably a combination of    -   (ii) water for injection;    -   (ii) sucrose;    -   (iii) Na₂HPO₄ and NaH₂PO₄;    -   (iv) diltiazem or nicardipine;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine,        as hereinbefore defined; together with lactobionic acid, KOH and        NaOH, glutathione and allopurinol and PEG, and any optional        additional components as herein defined; more preferably        comprises a combination of component classes given below, more        preferably of the specific type listed, and substantially in the        amount listed as follows, together with any optional additional        components as herein defined, when made up to volume in water:

Amount Component Type (mmol/L) impermeant sequestering Lactobionic 40-70anion component acid or lactobionate inorganic solute KOH & NaOH 40-70components components effective Glutathione and 3-5 against oxygen freeAllopurinol radicals pH buffer component Na₂HPO₄ & 40-70 (iii) NaH₂PO₄amino acid (vi) Glutamic acid; 2-20 or <20 or Glutamine such as 2-19.9;or 15-30 saccharide component Sucrose  50-150 (ii) anti-inflammatory (v)Salicylic acid; 0.025-0.5 or <0.5 or Aspirin such as 0.025- 0.49; or0.3-1.0 component with calcium Diltiazem 0.0005-1.0   transport blocking(iv) colloid component PEG (20,000 MW) 0.5-3.0

provided that if (v) is aspirin, (vi) is not glutamine;

and most preferably substantially in the amount listed as follows,together with any optional additional components as hereinbeforedefined, when made up to volume in water:

Amount Component Type (mmol/L) impermeant sequestering anion Lactobionic50 component acid inorganic solute component KOH 15 inorganic solutecomponent NaOH 35 component effective against Glutathione 3 oxygen freeradicals pH buffer component (iii) Na₂HPO₄ 26.45 pH buffer component(iii) NaH₂PO₄ 16.66 glutamic acid or glutamine(vi) Glutamic acid <20 or19.9; or glutamine or 20 saccharide component (ii) Sucrose 100 salicylicacid or aspirin (v) Salicylic acid or <0.5 or 0.49, aspirin or 0.5component effective against Allopurinol 0.4 oxygen free radicalscomponent with calcium transport Diltiazem 0.0022 blocking (iv) colloidcomponent PEG (20,000 MW) 1

provided that if (v) is aspirin, (vi) is not glutamine.

We have moreover surprisingly found that the effectiveness ofconstituents of a preservation solution according to the invention isimproved by the replacement of certain constituents with their principaldegradation products. Without being limited to this theory, it seemsthat the by-products of such degradation can exert unwanted toxiceffects during the use of the preservation solution both individuallyand in combination. For example acetic acid and ammonia form acetamidewhich is potentially damaging to the liver. By removing this toxiceffect, the effectiveness of the preservation solution is improved.

Accordingly, the finding according to the present invention is thatcertain substituents are essential for the preservation of the principlecell functions essential to all cell types and these have beenidentified as the component (i) to (vi) as herein defined. Whilst thissolution may be highly effective or satisfactory in preserving simpletissue or cellular systems, if it is desired to preserve organs or cellsystems requiring or providing unusual or more complex cell function, itis necessary to incorporate substituents specifically directed topreserve the requisite or provided function, whether this be muscular,electrical, specific membrane activity, energy supply and the like.

Thus, the invention particularly provides a preservation solution asherein defined for liver, kidney, small bowel and pancreas preservation.

The solution is suitably made up by methods as known in the art bysimple admixture under pharmacopoeially acceptable conditions.Preferably components are determined and incorporated in a desired molarconcentration.

It will be appreciated that variation may be specific or non-specific tothe effectiveness of the solution and that an amount of variation whichhas no effect on the performance of the fluid is considered within thescope of this invention. Selection of component type, requiring anamount of verification by routine experimentation, is considered withinthe scope of this invention.

In a further aspect of the invention there is provided a method for thepreparation of a preservation solution comprising adding components(i)-(vi) in sequence to water, together with any additional components,with the exception of the component with colloid osmotic properties andunstable components if any, e.g. aspirin or glutamine, and dissolvingthe mixture; adding the component with colloid osmotic properties, ifany, and making the solution nearly up to volume; and finally making upto volume to regulate pH, sterilising and cooling to 0-4° C. Thesolution may be stored if desired with subsequent addition of anyunstable components (e.g. aspirin and/or glutamine) immediately prior touse. The method according to this aspect of the invention may includepackaging the prepared solution for storage, preferably at reducedtemperature, more preferably in the range 0-12° C., most preferably 0-4°C. e.g. packaging the prepared solution in an anoxic condition and inthe absence of UV light.

In a further aspect there is provided a preservation solution obtainedby the method as herein described.

In embodiments herein there is provided a preservation solution obtainedby a method comprising adding components (i)-(vi) to water.

In a further aspect of the invention there is provided the use of apreservation solution as herein defined as a flush preservation solutionor a cold storage preservation solution; for the preservation of cellsin the absence of a blood supply, in particular to prevent damage toorgans, living tissues and cells. The solution is suited for use withsmall or large animal or mammalian, in particular human cells, tissuesand organs.

The use of the solution may be in transplantation including organs fromheart beating or non-heart beating donors, in surgery including anysituation of warm or cold ischaemia, whole limb or whole bodypreservation, in experimentation on living tissues, in culturing andpreserving engineered cells, tissues and organs and the like. Preferablythe solution is used as a flush solution brought into contact withcells, tissues or organs, limbs or the whole body via the vascularsystem, and optionally additionally serves as a preservation solutionfor storage of flushed cells, tissues and organs. The solution issuitable for extended preservation of the cells, tissues, organs in bothhypothermic static storage and with a hypothermic machine perfusionsystem.

Thus, the invention particularly provides a preservation solution asherein defined for liver, kidney, small bowel and pancreas preservation.

In a further aspect of the invention there is provided a method forflushing, preserving or flush preservation of cells, in particularliving cells, tissues or organs whereby the cells, tissue or organs arebrought into contact with a solution as herein defined.

According to this aspect of the invention there is provided a method forflushing, preserving or flush preservation of cells for simplehypothermic storage, in particular living cells, tissues or organswhereby the cells, tissue or organs are brought into contact with apreservation solution comprising:

-   -   (i) water for injection;    -   (ii) at least one saccharide, such as a monosaccharide,        disaccharide, trisaccharide, or polysaccharide;    -   (iii) at least one component with pH buffer properties;    -   (iv) optionally at least one component with calcium transport        blocking properties or an anti-calcium action activity;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine;    -   provided that acetamide is absent and/or if aspirin is present        glutamine is absent and if glutamine is present aspirin is        absent; more particularly wherein if (v) is aspirin (vi) is        glutamic acid, in free form or salt form, and if (vi) is        glutamine (v) is salicylic acid, in free form or salt form;    -   whereby the cells, tissue or organ are flushed with solution,        removed from the normal locus, cooled to temperatures normally        in the range between zero and 12° C. and stored.

According to this aspect of the invention if acetamide is absent, thenat least one of acetic acid and ammonia is absent.

According to one aspect of the invention the method comprises the flushpreservation of living cells, tissues or organs.

According to another aspect of the invention the method comprises coldstorage preservation of living cells, tissues or organs.

In a preferred method according to this aspect of the invention, aspirinis replaced by salicylic acid, in free form or in salt form, andglutamine is replaced by glutamic acid, in free form or in salt form,such that both aspirin and glutamine are substantially absent from thesolution, preferably acetamide is additionally absent and/or aceticacid, ammonia and acetamide are additionally absent, more particularlywherein (v) is salicylic acid, in free form or salt form, and (vi) isglutamic acid, in free form or salt form.

According to a further aspect of the invention there is provided amethod for the preservation of cells for simple hypothermic storage, inparticular living cells, tissues or organs whereby the cells, tissue ororgans are brought into contact with a preservation solution comprising:

-   -   (i) water for injection;    -   (ii) at least one saccharide, such as a monosaccharide,        disaccharide, trisaccharide, or polysaccharide;    -   (iii) at least one component with pH buffer properties;    -   (iv) optionally at least one component with calcium transport        blocking properties or an anti-calcium action activity;    -   (v) salicylic acid, in free form or in salt form, or aspirin;        and    -   (vi) glutamic acid, in free form or in salt form, or glutamine;        and    -   at least one component with cryoprotectant properties (i.e. a        cryoprotectant);    -   provided that acetamide is absent and/or at least one of acetic        acid and ammonia is additionally absent; more particularly        wherein if (v) is aspirin (vi) is glutamic acid, in free form or        salt form, and if (vi) is glutamine (v) is salicylic acid, in        free form or salt form;    -   whereby the cells, tissue or organ are flushed with solution,        removed from the normal locus, cooled to temperatures normally        in the range between −20° C. and 12° C. and stored.

According to this aspect of the invention if acetamide is absent, thenat least one of acetic acid and ammonia is absent.

The method may be for simple hypothermic storage, whereby the cells,tissue or organ are flushed with solution, removed from the normallocus, cooled, preferably to temperatures normally in the range betweenzero and 12° C. and stored. In addition, if a cryoprotectant is presentthe tissue or organ may be stored at a temperature of from about −20° C.to about 4° C. In addition, the solution may be actively flushed throughthe organ. We have found that cells, tissues or organs can be stored forextended periods exceeding those currently practised, for example,kidney and liver for periods of the order of 48 hours or more.Additionally, or alternatively, the method is for the preservation ofcells, particularly tissue or organs, whereby the cells, tissue ororgans have been flushed and brought into a hypothermic state and arecontacted with the preservation solution by immersion or perfusion.

Preferably, the method of the invention comprises administering to thecells, tissue, organ or to a donor a biologically effective amount ofthe solution of the invention, at an effective rate or in an effectiveconcentration to maintain or enhance function thereof. Preferably, themethod is a method for preserving certain cell, tissue or organfunction, for example cell metabolism, and/or for temporarily arrestingcertain functions, for example muscular activity, breakdown or excretionof essential cell components and the like, and/or excretory products forexample in the form of bile or urine and the like.

Ischaemia is the situation that results from the stopping of blood flowthrough an organ. The effects are due to lack of oxygen and nutrients;and accumulation of carbon dioxide and other waste products. It is moredamaging at body temperature than in the cold, which is why transplantorgans are flushed and cooled. Organ donors have frequently sufferedtrauma and the donor organ may therefore have been subjected to a periodof warm ischaemia as a result of the trauma. Adding a period of warmischaemia experimentally, prior to flush imitates this situation. It isan advantage that our solution provides protection from such warmischaemia.

Preferably, flush perfusion is carried out at a pressure of up to 300mmHg, more preferably in the range atmospheric to 200 mmHg, morepreferably in the range up to 160 mmHg, more preferably up to 100 mmHg,most preferably up to 50 mmHg.

The method according to this aspect of the invention comprises the useof the preservation solution as herein defined.

In a further aspect of the invention there is provided a kit of partscomprising a preservation solution having, in one part, components(i)-(vi) as herein defined, optionally together with, in one or morefurther parts, individual components selected from one or more of theadditional components as herein defined, for use in the preparation ofone or more solutions for specific purpose; and serving as a universalpreservation solution.

In embodiments a kit of parts herein comprises a preservation solutionhaving, in one part, components (i)-(vi) as herein defined, of which oneor more unstable components are absent and are provided separately, inone or more further parts, for addition immediately prior to use.

According to one aspect of the invention the kit of parts comprises aflush preservation solution having components (i)-(vi) as hereindefined.

According to another aspect of the invention the kit of parts comprisesa cold storage preservation solution having components (i)-(vi) asherein defined.

The invention is now illustrated in non-limiting manner with referenceto the examples and with reference to the accompanying figures, inwhich:

FIG. 1 illustrates the stability of aspirin in a preservation solution:HPLC methods were developed to measure the concentration of aspirin andsalicylic acid in the Prior Art solution. Aspirin is rapidly hydrolysedto salicylic acid and acetic acid during storage. After 8 weeks, none ofthe parent compound remains.

FIG. 2 illustrates serum creatinine following kidney preservation in anImproved Preservation solution (solution of the invention) versus aPrevious (prior art) Formulation. Lower serum creatinine illustratesreduced damage to organ.

FIG. 3 illustrates the ex vivo preservation of porcine kidney inpreservation solution of the invention up to 72 hours outside the body.The parenchyma is well preserved with normal glomerulus and tubules. H&Estaining, 40× magnification.

EXAMPLE 1

1.1 Flush Preservation Solutions Used in the Invention

The preservation solutions involved in this study are shown in theTables 1, 2, 3 and 4, amounts are given in mmol/L. Solutions (SOLS) weremade up from a flask half filled with water, to which any or all oflactobionate, KOH, sodium phosphate, glutamine or glutamic acid,sucrose, aspirin or salicylic acid, allopurinol and diltiazem were addedin sequence and dissolved. The colloid (PEG) was then added and thesolution made nearly up to volume. NaOH was added to set the pH. Thesolution was then made up to volume. All solutions were sterilized byfiltration and stored in glass bottles at 4° C. Reduced glutathione wasadded during preparation or immediately before use. Sources are asindicated above or as disclosed in WO 02/41696.

Comparative

Previous formulation is as disclosed and described in WO 02/41696 and asillustrated in Table 1 herein. UW is a standard original-commercialsolution (viaSpan/BELZER UW, DU PONT PHARMA).

Composition of Comparative Preservation Solutions

The composition of comparative preservation solutions is illustrated inTable 1:

TABLE 1 University of Prior Art Wisconsin Mmol/L Formulation Solution(Belzer) Lactobionic acid 50 105 (K-lactobionate) KOH 16 100 NaOH 35 KCl5 Adenosine 5 Glutathione 3 3 KHPO₄ (iii) 25 MgSO₄ 5 Na₂HPO₄ (iii) 26.45NaH₂PO₄ (iii) 16.66 Glutamine (vi) 20 Sucrose (ii) 100 Raffinose (ii) 30Aspirin (v) 0.5 Allopurinol 0.4 1 Nicardipine (iv) 0.005 PEG (20,000 MW)1 Pentastarch 5

Composition of the Cold Preservation Solutions of the Invention

The composition of the cold preservation solutions of the invention isillustrated in Table 2:

TABLE 2 Mmol/L SOL1.1 SOL1.2 SOL1.3 Lactobionic acid 50 50 50 KOH 5 5 5NaOH 45 45 45 Glutathione 3 3 3 Na₂HPO₄ 26.45 26.45 26.45 NaH₂PO₄ 16.6616.66 16.66 Glutamic acid (vi) 20 0 20 Glutamine (vi) 0 20 0 Sucrose 100100 100 Salicylic acid (v) 0.5 0.5 0 Aspirin (vi) 0 0 0.5 Allopurinol0.4 0.4 0.4 Diltiazem 0.022 0.022 0.022 PEG (20,000 MW) 1 1 1

Composition of the Cold Preservation Solutions of the Invention with (v)and/or (vi) in Reduced Amount

The composition of the cold preservation solutions of the invention with(v) and/or (vi) in reduced amount is illustrated in Table 3:

TABLE 3 Mmol/L SOL2.1 SOL2.2 SOL2.3 Lactobionic acid 50 50 50 KOH 5 5 5NaOH 45 45 45 Glutathione 3 3 3 Na₂HPO₄ 26.45 26.45 26.45 NaH₂PO₄ 16.6616.66 16.66 Glutamic acid (vi) 12 0 12 Glutamine (vi) 0 20 0 Sucrose 100100 100 Salicylic acid (v) 0.3 0.3 0 Aspirin (vi) 0 0 0.5 Allopurinol0.4 0.4 0.4 Diltiazem 0.022 0.022 0.022 PEG (20,000 MW) 1 1 1

Composition of the Cold Preservation Solutions of the Invention with (v)and/or (vi) in Reduced Amount

The composition of the cold preservation solutions of the invention with(v) and/or (vi) in reduced amount is illustrated in Table 4:

TABLE 4 Mmol/L SOL3.1 SOL3.2 SOL3.3 Lactobionic acid 50 50 50 KOH 5 5 5NaOH 45 45 45 Glutathione 3 3 3 Na₂HPO₄ 26.45 26.45 26.45 NaH₂PO₄ 16.6616.66 16.66 Glutamic acid (vi) 3 0 3 Glutamine (vi) 0 20 0 Sucrose 100100 100 Salicylic acid (v) 0.25 0.25 0 Aspirin (vi) 0 0 0.5 Allopurinol0.4 0.4 0.4 Diltiazem 0.022 0.022 0.022 PEG (20,000 MW) 1 1 1

EXAMPLE 2

The comparative solutions and the flush preservation solutions of theinvention were studied in the following systems.

2.1.1 Generation and Impact of Toxic Metabolites Produced by FlushPreservation Solutions Described in Prior Art

The flush preservation solution described in International patentapplication No. WO 02/41696 was found to generate, upon storage,metabolic products which have the potential to be toxic to thecells/tissues/organs under storage. As an example, FIG. 1 illustratesthat storage of the solution for 8 weeks results in 100% of the aspirinconverting to salicylic acid and the production of equimolar amounts ofdamaging acetic acid. In a solution which originally contains 0.5 mMaspirin, this results in the formation of 0.5 mM acetic acid, 30 mg/L.Acetic acid is a known irritant and is damaging to human tissues.

By way of further example, the presence of ammonia in the prior artsolution has been shown by colorimetric assay. The Ammonia Assay Kit(ab83360) provides a rapid, simple, sensitive, and reliable assaysuitable for research and high throughput assay of ammonia and ammonium.In the assay, ammonia and ammonium are converted to a product thatreacts with the OxiRed probe to generate color (λmax=570 nm) which canbe easily quantified by plate reader. The kit can detect 1 nmol (˜20 μM)of total ammonia and ammonium, which is much more sensitive thanmeasuring ammonia with a NADPH based assay. Using this protocol, it isdemonstrated that the Prior Art Solution contains approximately 2.6 mMammonia. This is many times greater than known toxicity of ammonia, 300μM is known to be toxic to human cells¹. In contrast, in the solution ofthe invention, ammonia is substantially absent, below the lower level ofquantification of the assay, less than 2 nmol. ¹ Heeneman et al, Journalof Immunological Methods (1993) 166, 1, p 85-91

Ammonia toxicity can occur by it causing a disturbance in the uptake ofpotassium by cells. This can cause deleterious effects in all organs andtissues; change in pH, membrane potential and metabolism². In vivo,ammonia would normally be detoxified by the hepatic urea cycle. However,this is unable to occur during the storage or use of the preservationsolution as the metabolic activity of the cells is intentionallyrepressed during cold preservation. This means that the ammonia in theprior art solution remains for the duration of preservation and there issignificant potential for cellular damage upon reperfusion. In asolution of the present invention, ammonia is absent and this damagecannot occur. ² Dasarathy, S., Mookerjee, R. P., Rackayova, V. et al.Metab Brain Dis (2017) 32: 529.https://doi.org/10.1007/s11011-016-9938-3

In a solution which originally contains 20 mM glutamine or approximately2.6 mM ammonia and 0.5 mM aspirin, or up to 0.5 mM acetic acid, thisresults in the formation of up to 0.5 mM acetamide, 30 mg/L. Acetamideis known to be damaging to human tissues.

2.1.2 Absence of Toxic Metabolites in Flush Preservation Solutions ofthe Invention

In solutions of the invention from which aspirin is absent, SOL 1.1, SOL1.2, SOL 2.1, SOL 2.2, SOL 3.1 and SOL 3.2, acetic acid is substantiallyabsent even after storage, below the lower level of quantification ofthe assay.

In further solutions of the invention in which aspirin is addedimmediately prior to use, acetic acid is substantially absent or is atreduced levels during the period of use.

In a solution of the invention from which glutamine is absent, SOL 1.1,SOL 1.3, SOL 2.1, SOL 2.3, SOL 3.1 and SOL 3.3, ammonia is substantiallyabsent even after storage, below the lower level of quantification ofthe assay, less than 2 nmol.

In further solutions of the invention in which glutamine is addedimmediately prior to use, ammonia is substantially absent or is atreduced levels during the period of use.

Further, in all solutions of the invention either aspirin or glutamineor both is absent, SOL 1.1, SOL 1.2, SOL 1.3, SOL 2.1, SOL 2.2, SOL 2.3,SOL 3.1, SOL 3.2 and SOL 3.3, and acetamide is substantially absent evenafter storage.

2.2 Porcine Transplantation Model

For true assessment of the quality of preservation solutions, it isnecessary to employ a whole animal model to assess the clinical outcomesof transplantation using the test solutions. The porcine model is mostappropriate for this. In brief the protocol involves retrieval of theorgan (liver, kidney, small bowel or pancreas), flush preservation inTest (Invention) or Control solutions (Comparison) and then implantation(allo- or auto-transplant depending on the organ under consideration).Organ function and overall health of the recipient is monitoredpost-surgery for a clinically appropriate length of time.

The following table gives an indication of the measurements used toassess the functionality of the transplanted organ in the recipientanimal. In addition, the overall health and wellbeing of the animal ismonitored (body weights, food consumption etc.). At the end of the studythe animal is sacrificed and key organs collected for histologicalanalysis.

Outcomes to be Measured Liver ALT AST LDH Bilirubin Prothrombin clottingtime Urea & electrolytes (Na and K) Kidney Urea and electrolytes (Na andK) Creatinine (used to calculate eGFR) Pancreas Urea and electrolytes(Na and K) Daily urine sample - qualitative assay for glucose Bloodglucose Blood amylase & lipase Glucose tolerance test

Creatinine is a key blood marker which directly relates to kidneyfunction. A functioning kidney will maintain a low concentration ofcreatinine; a high level indicates a kidney that is poorly functioning.Post-transplant, the serum creatinine is expected to rise and only fallsback to normal levels if/when kidney function is restored. The magnitudeof the rise reflects the magnitude of the preservation injury. The speedat which creatinine levels return to normal reflects how quickly thekidney returns to normal function; this itself is a key measure of howwell the organ was preserved. FIG. 2 illustrates a recent study withinwhich the preservation solution of the invention significantlyoutperforms the prior art solution; peak creatinine is significantlylower and kidney function returns to normal much quicker. This isevidence for much reduced tissue damage in the solution of theinvention.

Histology

Histology was performed of tissue samples collected from abdominalorgans (liver, kidney, pancreas and small intestine) of animals perfusedwith either University of Wisconsin (UW) (commercially available asBelzer UW® cold storage solution) or the preservation solution of theinvention for up to 72 hours. Standard comparator solutions such as UWare typically able to preserve tissue up to approximately 12-24 hours.Tissue samples stained by haematoxylin & eosin (FIG. 3) illustratenormal morphology and good preservation of samples by the preservationsolution of the invention for up to 72 hours. Better retention ofcytoplasmic contents is seen and nuclear ghosting was also present inthe UW group which was absent in the preservation solution of theinvention. Further nuclear ghosting is apparent in UW samples at 48 and72 hours which are absent with the preservation solution of theinvention samples. This is evidence that the preservation solution ofthe invention imparts much reduced cellular damage over extendedpreservation times.

1. A preservation solution for the preservation of cells, tissues and/ororgans, in the absence of a blood supply, said solution comprising: (i)water for injection; (ii) at least one saccharide; (iii) at least onecomponent with pH buffer properties; (iv) optionally at least onecomponent with calcium transport blocking properties or an anti-calciumaction activity; (v) salicylic acid, in free form or in salt form, oraspirin; and (vi) glutamic acid, in free form or in salt form, orglutamine; provided that acetamide is absent and/or if aspirin ispresent glutamine is absent and if glutamine is present aspirin isabsent, such that acetamide is not formed; wherein the solution is aflush preservation solution and/or a cold storage preservation solution.2. A preservation solution according to claim 1 wherein if (v) isaspirin, (vi) is glutamic acid, in free form or in salt form.
 3. Apreservation solution according to claim 1 wherein if (vi) is glutamine,(v) is salicylic acid, in free form or in salt form. 4-6. (canceled) 7.A preservation solution according to claim 1 wherein (v) is salicylicacid, in free form or in salt form; and (vi) is glutamic acid, in freeform or salt form, or glutamine; and aspirin is absent, such that aceticacid and/or acetamide are not formed.
 8. (canceled)
 9. A preservationsolution according to claim 1 wherein (v) is salicylic acid, in freeform or in salt form, or aspirin; and (vi) is glutamic acid, in freeform or salt form; and glutamine is absent, such that ammonia and/oracetamide are not formed.
 10. A preservation solution according to claim1 wherein aspirin and glutamine are absent. 11-14. (canceled)
 15. Apreservation solution according to claim 1 wherein the amount ofsalicylic acid, in free form or in salt form, is less than 0.5 mmol/L.16. (canceled)
 17. A preservation solution according to claim 9 whereinaspirin, if present, is in an amount of from about 0.3 mmol/L to about1.0 mmol/L.
 18. A preservation solution according to claim 1 wherein theamount of glutamic acid, in free form or in salt form, present is lessthan 20 mmol/L.
 19. (canceled)
 20. A preservation solution according toclaim 1 wherein glutamine, if present, is in an amount of from about 15mmol/L to about 30 mmol/L.
 21. A preservation solution according toclaim 1 wherein the saccharide is selected from one or more of fromsucrose, raffinose and mannitol.
 22. A preservation solution accordingto claim 21 wherein the saccharide is sucrose.
 23. A preservationsolution according to claim 1 wherein the pH buffer is selected from asodium phosphate buffer, a potassium phosphate buffer and the like suchas, Na₂HPO₄, NaH₂PO₄, K₂HPO₄, KH₂PO₄ and the like; and combinationsthereof.
 24. (canceled)
 25. A preservation solution according to claim 1wherein the calcium transport blocker or anti-calcium activity agent isselected from any known calcium transport or channel blocker, such asone or more of nicardipine, diltiazem, verapamil, nisoldipine,chlorpromazine or trifluorperazine.
 26. (canceled)
 27. A preservationsolution according to claim 1 wherein the preservation solution includesat least one anion that is largely impermeable into cells.
 28. Apreservation solution according to claim 27 wherein the anion that islargely impermeable into cells is an impermeant sequestering anion, suchas lactobionate or lactobionic acid.
 29. (canceled)
 30. A preservationsolution according to claim 1 wherein the preservation solution includesat least one component with colloid osmotic properties, such aspolyethylene glycol (PEG), succinylated gelatin (as in Gelofusine),Ficoll (a polysaccharide) or a starch product.
 31. (canceled)
 32. Apreservation solution according to claim 1 wherein the preservationsolution includes an inorganic or organic solute, such as an electrolyteincluding cations and/or anions, for example selected from Na⁺, K⁺, Cl⁻,OH⁻ and the like; and combinations thereof. 33-34. (canceled)
 35. Apreservation solution according to claim 1 wherein the preservationsolution includes at least one component with calcium chelatingproperties. 36-40. (canceled)
 41. A preservation solution according toclaim 1 wherein the preservation solution includes at least onecomponent that is effective against oxygen free radicals or theproduction of oxygen free radicals, such as allopurinol and reducedglutathione, or a combination thereof. 42-56. (canceled)
 57. Apreservation solution according to claim 1 wherein the solution has a pHin the range 6.5-7.8. 58-67. (canceled)
 68. A method for thepreservation of cells for simple hypothermic storage, in particularliving cells, tissues or organs whereby the cells, tissue or organs arebrought into contact with a preservation solution comprising: (i) waterfor injection; (ii) at least one saccharide; (iii) at least onecomponent with pH buffer properties; (iv) optionally at least onecomponent with calcium transport blocking properties or an anti-calciumaction activity; (v) salicylic acid, in free form or in salt form, oraspirin; and (vi) glutamic acid, in free form or in salt form, orglutamine; provided that acetamide is absent and/or if aspirin ispresent glutamine is absent and if glutamine is present aspirin isabsent; whereby the cells, tissue or organ are flushed with solution,removed from the normal locus, cooled to temperatures normally in therange between zero and 12° C. and stored. 69-100. (canceled)
 101. Apreservation solution for the preservation of cells, tissues and/ororgans, in the absence of a blood supply, said solution comprising: (i)water for injection; (ii) at least one saccharide; (iii) at least onecomponent with pH buffer properties; (iv) optionally at least onecomponent with calcium transport blocking properties or an anti-calciumaction activity; (v) salicylic acid, in free form or in salt form,and/or aspirin; and (vi) glutamic acid, in free form or in salt form,and/or glutamine; provided that if aspirin is present glutamine isabsent and if glutamine is present aspirin is absent and/or wherein ifaspirin and/or acetamide and/or acetic acid is present, the amount ofsalicylic acid present exceeds that which can be attributed todegradation from aspirin, for example as determined by the sum of theamounts of any acetic acid and of any acetamide present; and/or whereinif glutamine and/or acetamide and/or ammonia is present, the amount ofglutamic acid present exceeds that which can be attributed todegradation from glutamine, for example as determined by the sum of theamounts of any ammonia and of any acetamide present.
 102. (canceled)103. A preservation solution for the preservation of cells, tissuesand/or organs, in the absence of a blood supply, said solutioncomprising: (i) water for injection; (ii) sucrose; (iii) Na₂HPO₄ andNaH₂PO₄; (iv) diltiazem; (v) salicylic acid, in free form or in saltform, or aspirin; and (vi) glutamic acid, in free form or in salt form,or glutamine, provided that acetamide is absent and/or if aspirin ispresent glutamine is absent and if glutamine is present aspirin isabsent, such that acetamide is not formed; together with lactobionicacid, KOH and NaOH, glutathione and allopurinol and PEG, and anyoptional additional components; whereby the cells, tissue or organ areflushed with solution, removed from the normal locus, cooled totemperatures normally in the range between zero and 12° C. and stored.